Process for protein extraction

ABSTRACT

The invention includes a process for extracting a target protein from  E. coli  cells that includes lowering the pH of a whole  E. coli  cell solution to form an acidic solution, disrupting the cells to release the protein into the acidic solution, and separating the cellular debris from the released protein to obtain a protein product enriched in the heterologous target protein. The invention also includes addition of a solubility enhancer.

This application is a continuation of U.S. Utility application Ser. No.10/655,874, filed Sep. 5, 2003, which claims priority to U.S.Provisional Application Ser. No. 60/408,653, filed Sep. 6, 2002, thedisclosures of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

Biotechnological processes for producing pharmaceutical or diagnosticprotein products generally employ extraction and purification steps toobtain products of interest from a variety of sources. Sources of theproteins may include bacteria, yeast, and mammalian cell culture fluids,and extracts of naturally occurring tissues, for example.

Generally, the extraction and purification steps are numerous and entailvarious techniques. The type of product to be produced, its intendeduse, and other factors influence what steps are most appropriate, whatextent of purification is beneficial, and how the purification can beaccomplished. In general, the greater the desired product purity, themore steps that will be utilized in the process.

Standard protein purification protocols generally begin with disruptionof cells and a clarification step to remove cell and/or tissue debrisfrom the target protein. One common way of clarifying a solution is bycentrifugation. Efficiency of a centrifugation step depends on particlesize, density difference between particles and surrounding liquid,viscosity of the feedstock, and the like. For solutions obtained fromsmall cells, such as E. coli, the small particle size and high viscosityreduce the feed capacity during centrifugation and may interfere withthe clarification process. Thus, it is often recommended to combine acentrifugation step with microfiltration. Although microfiltration canalleviate some of the problems that are encountered, fouling of themicrofiltration membranes can be a further problem.

Each additional step in a protein purification process affects both thecost of the purification and the overall yield. Accordingly,manufacturers seek to obtain a desired product purity in the mosteconomical fashion. One way to lessen the production cost is to reducethe number of steps in a purification process. Alternatively, the stepsin existing processes can be modified or enhanced to reduce protein lossat each step.

One method that increases yield by eliminating process steps is expandedbed chromatography (“EBC”). EBC is a technique that utilizes anabsorbent in a stable fluidized bed. When EBC is used for purifyingproteins from solutions containing cell debris and/or tissue debris,prior centrifugation is not necessary. Although use of EBC eliminates aprocess step, both product loss and processing disadvantages can occurwith EBC. The EBC apparatus maintains adsorbent in the column with afrit, and can be fouled by cell debris in the solution applied to thecolumn. Fouling of the frit can decrease yield of product, increaseprocessing times, and in extreme cases, render the process unusable.

Therefore, there remains a need for processes and methods that caneffect higher purity protein purifications at lower costs.

SUMMARY OF THE INVENTION

The invention provides a process for extracting protein from E. colicells that includes lowering the pH of a solution containing eitherwhole or disrupted E. coli cells expressing a heterologous targetprotein, to form an acidic solution and separating cellular debris fromreleased protein to obtain a protein product enriched in theheterologous target protein.

The invention also provides a process for extracting protein from E.coli cells that includes lowering the pH of a solution containing wholeE. coli cells expressing a heterologous target protein to form an acidicsolution, disrupting the cells to release protein into the acidicsolution, and separating cellular debris from released protein to obtaina protein product enriched in the heterologous target protein.

The invention also provides a method as above that further includesaddition of at least one solubility enhancer to either a solution ofwhole or disrupted E. coli cells expressing a heterologous targetprotein.

The invention also provides a method for decreasing biomass-biomassinteractions, biomass-resin interactions, or a combination thereof of asolution of disrupted E. coli cells that includes lowering the pH of asolution containing whole E. coli cells expressing a heterologous targetprotein to form an acidic solution, and disrupting the cells to releaseprotein in the acid solution, wherein the biomass-biomass interactions,biomass-resin interactions, or a combination thereof of the disruptedcell solution is reduced as compared with a non-acidic solution of cellsthat is lowered in pH after cell disruption.

The invention further provides a method for altering a flocculent in asolution of disrupted E. coli cells that includes lowering the pH of asolution containing whole E. coli cells expressing a heterologous targetprotein to form an acid solution, and disrupting the cells to releaseprotein into the acidic solution, wherein moisture content of aflocculent in the released protein solution is greater when cells aredisrupted in an acidic solution as compared with a non-acidic solutionof cells that is lowered in pH after cell disruption.

The invention also provides a method for decreasing viscosity of asolution of disrupted E. coli cells that includes lowering the pH of asolution containing whole E. coli cells expressing a heterologous targetprotein to form an acidic solution, and disrupting the cells to releaseprotein in the acid solution, wherein the viscosity of the disruptedcell solution is reduced as compared with a non-acidic solution of cellsthat is lowered in pH after cell disruption.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph comparing back pressure (psi) as a function of timefor post-homogenization and pre-homogenization conditioned homogenatesduring EBC.

FIG. 2 is a photograph comparing fouling of a frit subjected topost-homogenization and pre-homogenization conditioned homogenates.

FIG. 3 is a photograph comparing pellets from centrifugedpost-homogenization and pre-homogenization conditioned solutions.

FIG. 4 is a graph of the flow rate (cm/hr) for EBC of apost-homogenization conditioned homogenate, a pre-homogenizationconditioned homogenate, and a buffer solution at pH ranging from 4.0 to6.5.

FIG. 5 is a photograph of the interactions between an EBC resin and thehomogenate with 30 mM and 45 mM MgSO₄.

FIG. 6 is a graph of the concentration (mg/mL) of Fab′2 in solutionswith variable concentrations of MgSO₄ and polyethyleneimine.

FIG. 7 is a bar graph of the capacity (g Apo2L/L resin) of the resin forApo2L protein at various concentrations of MgSO₄.

FIG. 8 is an SDS-PAGE gel of Anti-Tissue Factor homogenates at pH from 4to 7 that include either a solubility enhancer or various salts.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “about” applies to all numeric values, whetheror not explicitly indicated. The term “about” generally refers to arange of numbers that one would consider equivalent to the recited value(i.e., having the same function or result). In many instances, the term“about” may include numbers that are rounded to the nearest significantfigure.

Processes of the invention are generally directed towards extractingheterologous target proteins from E. coli cells. As used herein, theterm “heterologous target protein” refers to a recombinant protein thatis not normally expressed by the host cell, tissue, or species. Examplesof heterologous proteins include but are not limited to Apo2L/Trail, FabVEGF, and Anti-Tissue Factor antibody, produced in E. coli. It isgenerally understood that a process for extracting a heterologous targetprotein will result in a protein product that is enriched in theheterologous target protein, but may also contain other components.Examples of such other components include, but are not limited to,proteins normally expressed by the host cell or tissue, cellular debris,and cell broth for example.

As used herein, the term “biomass” refers at least to cellular debris.“Biomass-biomass interactions” generally refer to the interactionsbetween cellular debris that can lead to flocculation. “Biomass-resininteractions” generally refer to the interactions between cellulardebris and the resin of the EBC column that can cause the cellulardebris to attach to the column and may eventually contribute to foulingof the column.

As used herein, the phrase “disrupting the cells” refers to any processthat releases the contents of the cell to the solution containing thecell. Examples of methods of “disrupting the cells” include but are notlimited to homogenization, lysozyme, sonication, freeze-thaw, FrenchPress, and other chemical, mechanical, or physical cell disruptionmethods. The process step of “disrupting the cells” may be accomplishedwith one or more steps, for example, multiple steps using a 4 passhomogenization.

As used herein, the step of “separating the cellular debris from thereleased protein” can be accomplished by a variety of known techniques.Examples of such techniques include, but are not limited to,centrifugation, microfiltration, packed bed chromatography (“PBC”),expanded bed chromatography (“EBC”), or other types of columnchromatography, for example. The step of “separating the cellular debrisfrom the released protein” does not require a complete separation of allcellular debris.

Centrifugation can be utilized to separate the cellular debris from thereleased protein. Any centrifugation process can be used. Specificparameters of the centrifugation would depend, at least in part, onfactors such as the nature of the heterologous target protein (aminoacid sequence and charge, for example), and the nature of the host cellor tissue expressing the heterologous target protein. One example of acentrifugation process that can be used is a continuous disk stackcentrifuge.

EBC can be utilized to separate the cellular debris from releasedprotein. EBC is a single pass operation in which desired proteins arepurified from crude, particulate containing solution without the needfor clarification, initial purification, or concentration. Although notnecessary, EBC can be utilized with prior clarification, purification,or concentration steps, such as centrifugation, microfiltration, or thelike.

EBC utilizes an adsorbent bed that is suspended in an equilibrium causedby the balance between particle sedimentation velocity and upward liquidflow velocity. The adsorbent expands in the bed and creates a distancebetween the adsorbent particles (this distance corresponds to the voidvolume as that term is used in chromatography techniques), that allowsfor relatively unhindered passage of cells, cell debris, and otherparticulates that may be present in the solution.

Specific methods and protocols for using EBC to separate the releasedprotein from the cellular debris are generally known. See, for example,“Expanded Bed Adsorption, Principles and Methods”, Edition AB(18-1124-26), published by Amersham Pharmacia Biotech. Specificparameters of a useful protocol would depend, at least in part, on thenature of the heterologous target protein to be separated, the hostcells or tissue from which it is being separated, and other suchfactors.

A process of the invention includes a step of lowering the pH of asolution containing whole E. coli cells expressing a heterologous targetprotein to form an acidic solution. As used herein, “whole E. colicells” refers to E. coli cells that have not been disrupted. As usedherein, “acidic” means having a pH that is less than 7.0. The term“non-acidic” means having a pH that is 7.0 or greater.

In the process invention, the pH of the whole cell solution ispreferably lowered to a pH that is about 4.0-5.0, more preferably toabout 4.0-4.5. Alternatively, the pH may be lowered to no more than 4.0,and may even be lowered to about 2.0, depending on the nature of theheterologous target protein, for example.

Lowering the pH of the solution is generally accomplished by adding anacid. Any acid can be used. Preferred are those acids that have abuffering capacity at the lowered pH, for example, citric acid, aceticacid, and the like. In general, the acid will be added at aconcentration of about 50 to 100 mM, but higher or lower concentrationscan also be used.

It has surprisingly been discovered that lowering the pH of the wholecell solution to form an acidic solution before the cells are disruptedprovides specific process and product advantages. For example,viscosity, flocculation, flow rates, processing systems, and processingtimes can be improved when the pH is lowered prior to disrupting thecells. The yield and quality of the heterologous target protein can alsobe improved when the pH is lowered prior to disrupting the cells.

Generally, the benefits of lowering the pH increase as the pH isdecreased from 7. Lowering the pH before cell disruption may also serveto increase the purity of the protein product containing theheterologous protein. It is thought, but not relied upon, that loweringthe pH of the whole cell solution serves to enhance the purity of theprotein product by removing cell debris and whole cell contaminants fromthe solution after the cells have been disrupted. As the pH is loweredto an acidic pH, the purity of the heterologous target protein productgenerally increases. Furthermore, as the pH is lowered from pH 5 to 4,the purity of the target protein shows a more substantial increase inrelation to the drop from pH 6 to 5 for example.

The process of the invention, optionally and preferably, includesaddition of at least one solubility enhancer to either the whole cellsolution, or the acidic whole cell solution, or the disrupted cellsolution. Preferably, the at least one solubility enhancer is added tothe whole cell solution prior to or contemporaneously with the loweringof the pH, more preferably prior.

Generally, the solubility enhancer is added in an amount that iseffective to enhance the solubility of the heterologous target protein,and can depend, at least in part, on the nature of the heterologoustarget protein and the homogenate or the whole cell solution. Generally,increased amounts of solubility enhancer will increase the solubility ofthe heterologous target protein.

Solubility enhancers are preferably molecules that include a divalentcation, such as magnesium (Mg⁺²), calcium (Ca⁺²), and the like.Preferred solubility enhancers for use in a process of the inventioninclude, but are not limited to magnesium sulfate (MgSO₄), magnesiumchloride (MgCl₂), calcium sulfate (CaSO₄), and calcium chloride (CaCl₂).

The solubility enhancers are generally added in aqueous form, in anamount that results in a final concentration of from about 10 mM toabout 150 mM, preferably from about 30 mM to about 120 mM.

Polyethyleneimine (“PEI”) can also function as a solubility enhancer ina process of the invention. Generally, the PEI is added to the solutionin an aqueous form and in an amount that results in a finalconcentration of about 0.1% to about 0.5% vol/vol of a 50% wt/volsolution, preferably about 0.2% to about 0.3%.

At least one solubility enhancer can be used alone, or in combination,and can be added to the process at any step, but are preferably addedprior to cell disruption.

The invention also provides methods of decreasing the biomass-biomassinteractions, biomass-resin interactions, or a combination thereof in asolution of disrupted E. coli cells that includes lowering the pH of thesolution containing whole E. coli cells expressing a heterologous targetprotein to form an acidic solution, and disrupting the cells to releaseprotein, wherein the biomass-biomass interactions, biomass-resininteractions, or a combination thereof of the disrupted cell solution isreduced as compared with a non-acidic solution of cells that is loweredin pH after cell disruption.

The level of the biomass-biomass interactions, biomass-resininteractions, or a combination thereof of a solution can also bemonitored based on its effects on the process that the solution isundergoing, such as EBC, PBC, centrifugation, or microfiltration forexample. These effects include but are not limited to aggregation ofcell debris (also referred to as formation of homogenate or biomass orflocculation), flow rate through a column, fouling of a frit, fouling ofthe continuous disk-stack centrifuge, backpressure on an EBC column, orcolumn inlet screen fouling, and the like.

The invention also provides a method for decreasing viscosity of asolution of disrupted E. coli cells that includes lowering the pH of asolution containing whole E. coli cells expressing a heterologous targetprotein to form an acidic solution, and disrupting the cells to releaseprotein in the acid solution, wherein the viscosity of the disruptedcell solution is reduced as compared with a non-acidic solution of cellsthat is lowered in pH after cell disruption.

The viscosity may be measured by any method known to those of ordinaryskill in the art, having read this specification. Examples of devicesfor measuring viscosity include, but are not limited to the Engler,Saybolt, and Redwood viscometers. All of the exemplary viscometersindicate the viscosity by the rate of flow of the test liquid through anorifice of standard diameter or the flow rate of a metal ball though acolumn of the liquid. Other types of viscometers utilize the speed of arotating spindle or vane immersed in the test liquid. Other exemplarytypes of viscometers include Brookfield and Krebs-Stormer devices.

The invention also provides methods of altering a flocculent in asolution of disrupted E. coli cells that includes lowering the pH of asolution containing whole E. coli cells expressing heterologous targetprotein to form an acidic solution, and disrupting the cells to releaseprotein, wherein moisture content of a flocculent in the releasedprotein solution is greater when cells are disrupted in an acidicsolution as compared with a non-acidic solution of cells that is loweredin pH after cell disruption.

As used herein, the phrase “altering the flocculent” includes changesthat increase moisture content, lessen tendency for flocculantaggregation, and the like.

Another method of monitoring flocculent in a homogenate is to monitorfouling of a continuous disk-stack centrifuge. In one embodiment, thiscan be measured by measuring turbidity. Turbidity can be measured in NTU(National Turbidity Units).

EXAMPLES

The following Examples are offered by way of illustration and are notintended to limit the invention.

Example 1 Conditioning E. coli Cells Prior to Disruption Alters ProteinFlocculation

E. coli cells containing a transgene expressing anti-VEGF antibodyfragment (Rhufab V2, Chen et. al., 1999 J. Mol. Biol. 293: 865-881) werepre-conditioned before homogenization by lowering the pH of the cellsolution to pH 4.0 via addition of 60 mM citric acid. A solubilityenhancer, MgSO₄, was also added to the cell solution, to a concentrationof approximately 120 mM. The cells were disrupted by a 4 passhomogenization with a Model HC-8000/3A homogenizer (Microfluidics Corp.,Newton, Mass.) at 8000 psig, 2-8° C. After homogenization, 5 volumes ofwater were added to the homogenate, and the homogenate was passedthrough an EBC inlet screen (Multilayered SS316L, 100 μm, 1.8 mm thick,2.5 cm diameter) (G.Bopp & Co., Zurich, Switzerland), with a flow rateof 25 ml/minute (300 cm/hr).

Control cells were similarly disrupted by a 4 pass homogenization, butwith post-homogenization conditioning at pH 4.0 and without added MgSO₄.Acid was added to the homogenized cells, to pH 4.0 as well as MgSO₄ (120mM) and 2 volumes of water. The resulting material was centrifuged topellet insoluble material. The control supernatant was applied to theEBC inlet screen, described above. The results, shown in FIGS. 1 and 2and in the table below, demonstrate that the pre-conditioning of E. colicells at pH 4.0 and in the presence of MgSO₄ reduced biomass-biomassinteraction. This reduction is demonstrated by reduced flocculation ofthe protein solution, as seen in the difference in backpressure, 0 psigfor the pre-conditioned homogenate versus 11 psig for thepost-conditioned homogenate (see Table 1, as well as FIG. 1). FIG. 1demonstrates the pressure as a function of time for pre-homogenizationconditioning and post-homogenization conditioning. TABLE 1 Backpressureincrease on EBC outlet screen (L of Homogenate)/cm² psigPre-Conditioning >4.3 0 Post-Conditioning 1.7 11

FIG. 2 demonstrates that the flocculated material found in thepost-conditioning control is absent from the screen of thepre-conditioned sample. These results evidence a reduction of thebiomass-biomass interactions when pH is reduced prior to disruptingcells to release protein.

Example 2 Conditioning of E. coli Cells Prior to Disruption DecreasesFouling of Continuous Disk-Stack Centrifuge

A pBR322-based plasmid vector was used to express a transgene expressingFab VEGF (Chen et al., 1999, J. Mol. Bio., 293: 865-881) in E. colicells (strain: 60E4, genotype: W3110 DfhuA DphoA DhtrA DompT DptrilvG2096 DrhaR DfucP). The vector had a rop deletion to produce a highercopy number, and used the phoA promoter for transcription. Each genecontained a heat stable enterotoxin II signal sequence prior to theantibody gene for export to the periplasm. Both tetracycline andampicillin resistances were intact on the plasmid. The fermentationswere done on a 10 L scale with a fixed level of phosphate in the mediasuch that the cells depleted phosphate as they grew, leading toinduction of the phoA promoter. The E. coli cells werepre-homogenization conditioned by lowering the pH of the cell solutionto pH 4.0 via addition of 60 mM citric acid. A solubility enhancer,MgSO₄, was also added to the cell solution, to a concentration ofapproximately 60 mM. The cells were disrupted by a 4 pass homogenizationwith a Model HC-8000/3A homogenizer (Microfluids, Inc.) at 8000 psig,2-8° C. After homogenization, 2 volumes of water were added to thehomogenate, and the homogenate was centrifuged in a continuousdisk-stack centrifuge (Model PX205, Alfa Laval, Inc., Richmond, Va.),with a flow rate of 1.0 L/minute. The shoot rate was set to shoot onceevery time 60% of the bowl volume was reached.

Control cells were similarly disrupted by a 4 pass homogenization, butwith post-homogenization conditioning. 60 mM citric acid was added tothe homogenized cells, to pH 4.0 as well as MgSO₄ (60 mM) and 2 volumesof water. The resulting material was centrifuged to pellet insolublematerial.

Fouling of the continuous disk centrifuge was measured as an increase inturbidity (NTU) of the centrifuged protein homogenate. The data aresummarized in Table 2, and demonstrate reduced fouling (solutionturbidity) of the centrifuge when cells are pre-conditioned by loweringpH and adding a solubility enhancer prior to disruption. TABLE 2Turbidity Vol. Homogenate NTU Pre-homogenization >1000 L <50Post-homogenization  <100 L >100

FIG. 3 shows the pellet from a centrifugation in the continuousdisk-stack centrifuge for pre-homogenization conditioning andpost-homogenization conditioning. As can be seen in FIG. 3, theconsistency of the flocculent is very different. The pellet from thepre-homogenization conditioning step appears to have more moisturecontent and produces a smoother “solid” than does the pellet from thepost-homogenization conditioning step. These results evidence areduction of the biomass-biomass interactions when cells arepre-conditioned prior to disruption.

Example 3 Conditioning of E. coli Cells Prior to Disruption IncreasesFlow Rate on the EBC Column

To analyze the effects of pre-conditioning on flow rate, Fab VEGFhomogenate obtained from pre-conditioned cells, prepared as describedabove in Example 1 and with control, post-conditioned homogenate wereeach added to SPXL Streamline (Amersham Bioscience, Inc. Piscataway,N.J.) EBC columns (2.6 cm diameter×100 cm high) with a resin settled bedheight of 30 cm, and an expanded bed height of 90 cm. The EBC columnswere each equipped with an EBC inlet screen (Multilayered SS316L, 100μm, 1.8 mm thick, 2.5 cm diameter). The flow rate of the homogenatesthrough the columns were compared to each other using buffer as acontrol. The pre- and post-homogenization conditioning samples wereprepared at conditioning pH 4.5, 5.0, 5.5, 6.0, and 6.5.

The results are depicted in FIG. 4, and demonstrate thatpre-homogenization conditioning resulted in an increased flow ratethrough the EBC column. The flow rate also increased as the pH islowered. These results evidence a reduction of the biomass-biomassinteractions as well as a reduction of the biomass-EBC resininteractions, and indicate better flow rates obtained withpre-conditioning.

Example 4 Effect of Concentration of Solubility Enhancer onBiomass-Resin Interactions

E. coli cells (strain: 43E7, genotype: W3110 DfhuA phoADE15D(argF-lac)169 ptr3 degP41 DompT (DnmpC-fepE) ilvG2096) containing atransgene expressing Apo2L (Pitlie et al., 1996, J. Bio. Chem., 271:12687-12690) were used to express protein. A pBR22-based plasmid vectorwith a phoA promoter driving transcription was used for the expression.The plasmid contained 3 tRNA genes for enhanced production and had bothtetracycline and ampicillin resistances intact. The cells were disruptedby a 4 pass homogenization with a Model HC-8000/3A homogenizer(Microfluids, Inc.) at 8000 psig, 2-8° C.

The homogenate was conditioned by addition of a solubility enhancer,MgSO₄, to a concentration of approximately 45 mM or 30 mM. Two (2)volumes of water were added, and the pH of the homogenate was adjustedto pH 6.5 via addition of 60 mM citric acid. The conditioned homogenatewas run through an EBC column (2.5 cm diameter×210 cm high) containingSP Streamline (Amersham Bioscience, Inc.) with a resin settled bedheight of 65 cm, and an expanded bed height of 195 cm. The EBC columnwas equipped with an EBC inlet screen (Multilayered SS316L, 100 μm, 1.8mm thick, 2.5 cm diameter) (Amersham Bioscience, Inc). The flow rate inthe column was 200 cm/hour.

FIG. 5 shows photographic images of the resin taken from the top screenafter the storage phase demonstrates a difference in the resin-biomassinteractions in the two homogenate solutions in the EBC columnconditioned with differing amounts of MgSO₄. The homogenate conditionedwith the higher concentration of MgSO₄ (45 mM) showed less resin-biomassinteractions than that conditioned with 30 mM MgSO₄, which showed muchgreater aggregation of the resin.

Example 5 Effect of Concentration of Solubility Enhancer on ProteinSolubility

E. coli cells (strain: 60H4, genotype: W3110 DfhuA phoADE15D(argF-lac)169 degP41 deoC Dprc spr DmanA) containing a transgeneexpressing Anti-Tissue Factor antibody (Presta et al., 2001, Thromb.Haemost., 85: 379-89) were disrupted by a 4 pass homogenization with aModel HC-8000/3A homogenizer (Microfluids, Inc.) at 8000 psig, 2-8° C.The homogenate was conditioned by addition of two solubility enhancers,PEI at various concentrations and MgSO₄, to a concentration of 0 mM, 10mM, 20 mM, 30 mM, 40 mM, or 50 mM. The pH of the solution was adjustedto pH 4.0 via addition of citric acid and 2 volumes of water were addedto the adjusted solution. The homogenate was centrifuged to removesolids, and the supernatant was added to a Porous-G affinity column(Perceptives Biosystems). Protein was eluted from the column with low pHbuffer and quantitated.

FIG. 6 shows the concentration of Anti-Tissue Factor antibody protein inthe supernatant for each of the test samples. These results generallyshow that as the amount of the solubility enhancer, MgSO₄ or PEI, wasincreased, more protein was obtained.

Example 6 Effect of Concentration of Solubility Enhancer on EBC ResinCapacity

E. coli cells containing a transgene expressing Apo2L /TRAIL (Ashkenaziet al., 1999, J. Clin. Invest., 104: 155-162) were disrupted by a 4 passhomogenization with a Model HC-8000/3A homogenizer (Microfluids, Inc.)at 8000 psig, 2-8° C. After homogenization, the homogenate wasconditioned through addition of a solubility enhancer, MgSO₄, to aconcentration of 10 mM, 20 mM, or 40 mM. A control homogenate containedno MgSO₄. Next, the pH of the homogenate was adjusted to pH 6.5 viaaddition of 60 mM citric acid, and 2 volumes of water were added to theadjusted solution. The conditioned homogenate was then mixed with 1 mLof EBC resin, SP Streamline (Amersham Bioscience, Inc.) in a 15 mLconical plastic screw cap tube, in excess of the conditioned homogenate.

The capacity of the resin for each of the conditioned homogenatesolutions was determined, and the data is shown in FIG. 7. The presenceof the solubility enhancer, MgSO₄, increased the capacity of the resinfor Apo2L at 10 mM and 20 mM. However, as the concentration of the MgSO₄continued to increase (40 mM), the resin capacity decreased. Thisdecrease may be due to the increased conductivity of the solution withincreased concentrations of MgSO₄.

Example 7 Effect of pH Adjustment on Protein Purity

E. coli cells containing a transgene expressing Anti-Tissue Factorantibody were disrupted by a 4 pass homogenization with a ModelHC-8000/3A homogenizer (Microfluids, Inc.) at 8000 psig, 2-8° C. Thehomogenate was conditioned through addition of PEI at 0.2%. Varioussalts were also added, including the solubility enhancer, MgSO₄ at 100mM, NaCl at 250 mM, and Na₂SO₄ at 100 mM. The pH of the solution wasadjusted to pH 4.0, 5.0, 6.0, or 7.0 by addition of citric acid and 2volumes of water were added. The homogenate was centrifuged and thesupernatant was subjected to SDS-PAGE analysis at 10% Bis-Tris/MOPS(Novex, Inc.) with Coomassie Blue staining.

FIG. 8 is an electrophoretic gel showing the resulting protein products.The gel demonstrates that as the pH of the cell homogenate was decreasedfrom 7.0 to 4.0, the amount of contaminating protein (that is, proteinother than the heterologous protein, Anti-Tissue Factor antibody)present in the homogenate decreased. A dramatic enrichment of thedesired heterologous protein product in the E coli cell homogenateoccurred when the pH was dropped from 5.0 to 4.0.

Example 8 Effect of pH Adjustment on Protein Purity

E. coli cells containing a transgene expressing Anti-Tissue Factor weredisrupted by a 4 pass homogenization with a Model HC-8000/3A homogenizer(Microfluids, Inc.) at 8000 psig, 2-8° C. The homogenate was conditionedby addition of two solubility enhancers, PEI at 0.2% and MgSO₄, at 100mM. The pH of the solution was adjusted to pH 4.0, 5.0, 6.0, or 7.0 byaddition of citric acid and 2 volumes of water were added. Thehomogenate was centrifuged and the supernatant was subjected to HPLCusing a Protein G column (Perceptive Biosystems, Inc.) with a Bradfordstaining reagent (Pierce Chemical Co., Rockford, Ill.). The data areshown below in Table 3, and demonstrate a higher fold increase inprotein purity when the protein was produced from a homogenateconditioned at acidic pH. TABLE 3 Purity Increase pH Brad (g/L) ProG(g/L) ProG/Brad (pH/pH 7) 4.0 8.5 2.12 0.250 5.0 fold 5.0 25.3 2.280.090 1.7 fold 6.0 36.9 2.35 0.064 1.2 fold 7.0 45.0 2.33 0.052 —

Example 9 Comparison of EBC Using a Solubility Enhancer and PBC WithoutSolubility Enhancer

Packed bed chromatography purification was carried out as follows. E.coli cells containing a transgene expressing Apo2L/Trail were subjectedto a 4 pass homogenization with a Model HC-8000/3A homogenizer(Microfluids, Inc.) at 8000 psig, 2-8° C. After homogenization, twovolumes of water were added and polyethyleneimine was also added toresult in a final concentration of 0.3% by weight. The homogenatesolution was then centrifuged in a continuous disk-stack centrifuge(Model PX205, Alfa Laval, Inc.), with a flow rate of 1.0 L/minute. Thesupernatant was purified on a SP Sepharose FF column (2.5×20cm)(Amersham Biosciences).

The EBC purification was carried out as follows. E. coli cellscontaining a transgene expressing Apo2L/Trail were subjected to a 4 passhomogenization with a Model HC-8000/3A homogenizer (Microfluids, Inc.)at 8000 psig, 2-8° C. After homogenization magnesium sulfate (MgSO₄) wasadded to a final concentration of 30 mM, two volumes of water wereadded, and the pH of the solution was adjusted to pH 6.5. The homogenatewas then subjected to EBC with a SP Streamline (Amersham Bioscience,Inc.) adsorbent in a (2.5×65) cm column and a (2.5×14 cm) column.

Table 4 below compares the recovery of Apo2L/Trail from the threeprocesses. TABLE 4 Percent Recovery Technique Extraction Cent. Chrom.Overall Packed (SPFF) 2.5 cm diameter 84% 88% 95% 70% Expanded (SP Str.)2.5 cm diam. 95% 95% Expanded (SP Str.) 14 cm diam. 94% 94%

The EBC protocols utilized above resulted in the same flow rate (200cm/hour) and the same load volume (2 volumes of water) as the PBCprotocol; therefore, there was no loss in overall load process time,which was 4.5 hours. The addition of MgSO₄ as a solubility enhancerserved to enhance the solubility of the Apo2L/Trail as the pH wasdecreased from 7.5 to 6.5, thereby increasing the EBC resin capacity.The MgSO₄ reduced the homogenate-resin interactions such that the lowdensity resin aggregate formation was minimized during the EBC loadphase, thereby increasing protein recovery.

Example 10 Comparison of Packed Bed Chromatography and Expanded BedChromatography with Homogenate Conditioning

The packed bed chromatography (PBC) purification was carried out asfollows. E. coli cells containing a transgene expressing Apo2L/Trailwere subjected to a 4 pass homogenization with a Model HC-8000/3Ahomogenizer (Microfluids, Inc.) at 8000 psig, 2-8° C. Afterhomogenization, two volumes of water were added and polyethyleneiminewas added to a final concentration of 0.3% by weight. The solution wasthen centrifuged in a continuous disk-stack centrifuge (Model PX205,Alfa Laval, Inc.), with a flow rate of 1.0 L/minute. The supernatant waspurified on a SP Sepharose FF column (2.5×20) cm (Amersham Biosciences).

The expanded bed chromatography (EBC) purification was carried out asfollows. E. coli cells containing a transgene expressing Apo2L/Trailwere subjected to a 4 pass homogenization with a Model HC-8000/3Ahomogenizer (Microfluids, Inc.) at 8000 psig, 2-8° C. MgSO₄ was added tothe homogenate to obtain a final concentration of 30 mM MgSO₄. The pH ofthe solution was also adjusted to pH 6.5 and five volumes of water wereadded. The homogenate was then subjected to EBC with a SP Streamlineadsorbent (Amersham Biosciences, Inc.) in a (2.5×30) cm column and a(2.5×14)cm column.

Table 5 below compares the recovery of FAB V2 (AMD) from the threeprocesses. TABLE 5 Percent Recovery Technique Extraction Cent. ChromOverall Packed (SPFF) 2.5 cm diameter 90% 86% 97% 75% Expanded (SP Str.)2.5 cm diam. 96% 96% Expanded (SP Str.) 14 cm diam. 97% 97%

Although the EBC protocol utilized above resulted in a total load timethat was 4 times longer than that of the PBC protocol (24 hours versus 6hours), protein recovery was greatly increased using EBC. MgSO₄ servedto reduce both homogenate-homogenate and homogenate-resin interactions,and thereby enhanced the processing parameters of the EBC protocol.Adjustment of the pH before homogenization (pre-conditioning) also aidedprocessing by reducing homogenate-homogenate interactions, furtherincreasing protein yields (data not shown).

The above specification, examples and data provide a completedescription of the manufacture and use of the composition of theinvention. Since many embodiments of the invention can be made withoutdeparting from the spirit and scope of the invention, the inventionresides in the claims hereinafter appended.

The specification includes references to numerous patent and literaturecitations. Each is hereby incorporated by reference for all purposes, asif fully set forth.

1. A method for decreasing biomass-biomass interactions, biomass-resin interactions, or a combination thereof, of a solution of disrupted E. coli cells, comprising: a) lowering the pH of a solution containing whole E. coli cells expressing a heterologous target protein to form an acidic cell solution; b) adding at least one solubility enhancer to the solution containing the E. coli cells; and c) disrupting the cells to release protein into the acidic solution; wherein the biomass-biomass interactions, biomass-resin interactions, or a combination thereof of the disrupted cell solution is reduced as compared with a solution of cells disrupted at a non-acidic pH.
 2. The method of claim 1, wherein the moisture content of a flocculent in the released protein solution is greater when cells are disrupted in an acidic solution as compared with a non-acidic solution.
 3. The method of claim 1, wherein the pH is lowered to about 4.0-5.0.
 4. The method of claim 3, wherein the pH is lowered to about 4.0-4.5.
 5. The method of claim 4, wherein the pH is lowered to no more than 4.0.
 6. The method of claim 1, wherein the at least one solubility enhancer comprises a divalent cation.
 7. The method of claim 6, wherein the divalent cation comprises magnesium or calcium.
 8. The method of claim 1, wherein the at least one solubility enhancer is PEI.
 9. The method of claim 1, wherein the at least one solubility enhancer comprises a divalent cation and PEI.
 10. The method of claim 6, wherein the divalent cation is added at a concentration of about 10 mM to about 150 mM.
 11. The method of claim 8, wherein the PEI is added at a concentration of about 0.2% to about 0.3% vol/vol of a 50% wt/vol solution. 